The herpes simplex virus (HSV) DNA polymerase (pol) gene affords a unique model system amenable to both molecular and genetic approaches for studies of eukaryotic genes involved in DNA replication. The overall aim of these studies is to understand the pol gene in detail in terms of both gene structure and gene function. With the aid of cloned DNA fragments, the pol gene's nucleotide sequence will be determined and its RNA transcripts identified and mapped. The pol gene's polypeptide products will be defined and mapped by In Vitro translation of RNA hybridizing to the gene and by examination of the components of purified polymerase, with the aid of mutants which specify pol polypeptides with altered electrophoretic mobilities. Pol gene function will be explored by utilizing mutants which exhibit altered sensitivities to a variety of anti-viral drugs and the specific Alpha-polymerase inhibitor, aphidicolin. These mutants affect different functional properties of the polymerase, such as substrate recognition, in different ways. Domains governing these functions will be located, and amino acid alterations which affect them, identified, by cloning, mapping, and sequencing mutant DNA's. Similarly, pol mutants which affect the mutation rate of HSV will be mapped and sequenced to identify domains of the pol gene which govern replication fidelity. Thymidine kinase (TK) genes from TK-deficient mutants which arise spontaneously from wild-type virus and derivatives with altered mutation rates will be analyzed by DNA sequencing to determine if there is any specificity in the types of mutations generated by these strains. Wild-type HSV mutates at a high rate; these studies should shed light on this phenomenon. HSV is a serious health problem with no known cure. Since HSV polymerase is a target for several licensed and promising antiviral drugs, information from these studies should aid drug development. Similarly, HSV polymerase provides a model for human Alpha-polymerase which is a potential target for antitumor drugs. Moreover, the pol gene is a major factor in the high mutation rate of HSV which not only contributes to the development of resistance to antiviral drugs, but which may also be important for the life-cycle and disease causing properties of the virus. Thus, the proposed studies address issues regarding eukaryotic DNA replication and issues of direct health relevance.